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1999
Cross-linking between cytochrome c3 and flavodoxin from Desulfovibrio gigas, Correia, C., Monzani E., Moura I., Lampreia J., and Moura J. J. , Biochem Biophys Res Commun, Mar 16, Volume 256, Number 2, p.367-71, (1999) AbstractWebsite

Tetraheme cytochrome c3 (13 kDa) and flavodoxin (16 kDa), are small electron transfer proteins that have been used to mimic, in vitro, part of the electron-transfer chain that operates between substract electron donors and respiratory electron acceptors partners in Desulfovibrio species (Palma, N., Moura, I., LeGall, J., Van Beeumen, J., Wampler, J., Moura, J. J. G. (1994) Biochemistry 33, 6394-6407). The electron transfer between these two proteins is believed to occur through the formation of a specific complex where electrostatic interaction is the main driving force (Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P.K. and Wampler, J.E. (1988) Biochemistry 27, 2444-2450, Stewart, D., LeGall, J., Moura, I., Moura, J.J.G., Peck, H.D., Xavier, A.V., Weiner, P., Wampler, J. (1989) Eur. J. Biochem. 185, 695-700). In order to obtain structural information of the pre-complex, a covalent complex between the two proteins was prepared. A water-soluble carbodiimide [EDC (1-ethyl-3(3 dimethylaminopropyl) carbodiimide hydrochloride] was used for the cross linking reaction. The reaction was optimized varying a wide number of experimental parameters such as ionic strength, protein and cross linker concentration, and utilization of different cross linkers and reaction time between the crosslinker and proteins.

Biochemical and spectroscopic characterization of overexpressed fuscoredoxin from Escherichia coli, Pereira, A. S., Tavares P., Krebs C., Huynh B. H., Rusnak F., Moura I., and Moura J. J. , Biochem Biophys Res Commun, Jun 24, Volume 260, Number 1, p.209-15, (1999) AbstractWebsite

Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfido-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mossbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data.

MAD structure of Pseudomonas nautica dimeric cytochrome c552 mimicks the c4 Dihemic cytochrome domain association, Brown, K., Nurizzo D., Besson S., Shepard W., Moura J., Moura I., Tegoni M., and Cambillau C. , J Mol Biol, Jun 18, Volume 289, Number 4, p.1017-28, (1999) AbstractWebsite

The monohemic cytochrome c552from Pseudomonas nautica (c552-Pn) is thought to be the electron donor to cytochrome cd1, the so-called nitrite reductase (NiR). It shows as high levels of activity and affinity for the P. nautica NiR (NiR-Pn), as the Pseudomonas aeruginosa enzyme (NiR-Pa). Since cytochrome c552is by far the most abundant electron carrier in the periplasm, it is probably involved in numerous other reactions. Its sequence is related to that of the c type cytochromes, but resembles that of the dihemic c4cytochromes even more closely. The three-dimensional structure of P. nautica cytochrome c552has been solved to 2.2 A resolution using the multiple wavelength anomalous dispersion (MAD) technique, taking advantage of the presence of the eight Fe heme ions in the asymmetric unit. Density modification procedures involving 4-fold non-crystallographic averaging yielded a model with an R -factor value of 17.8 % (Rfree=20.8 %). Cytochrome c552forms a tight dimer in the crystal, and the dimer interface area amounts to 19% of the total cytochrome surface area. Four tighly packed dimers form the eight molecules of the asymmetric unit. The c552dimer is superimposable on each domain of the monomeric cytochrome c4from Pseudomomas stutzeri (c4-Ps), a dihemic cytochrome, and on the dihemic c domain of flavocytochrome c of Chromatium vinosum (Fcd-Cv). The interacting residues which form the dimer are both similar in character and position, which is also true for the propionates. The dimer observed in the crystal also exists in solution. It has been hypothesised that the dihemic c4-Ps may have evolved via monohemic cytochrome c gene duplication followed by evolutionary divergence and the adjunction of a connecting linker. In this process, our dimeric c552structure might be said to constitute a "living fossile" occurring in the course of evolution between the formation of the dimer and the gene duplication and fusion. The availability of the structure of the cytochrome c552-Pn and that of NiR from P. aeruginosa made it possible to identify putative surface patches at which the docking of c552to NiR-Pn may occur.

Structural studies by X-ray diffraction on metal substituted desulforedoxin, a rubredoxin-type protein, Archer, M., Carvalho A. L., Teixeira S., Moura I., Moura J. J., Rusnak F., and Romao M. J. , Protein Sci, Jul, Volume 8, Number 7, p.1536-45, (1999) AbstractWebsite

Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 x 36 amino acids) protein. Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3+, Cd2+, Hg2+, and Ni2+ salts. The In3+ and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory.

Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods, Dias, J. M., Than M. E., Humm A., Huber R., Bourenkov G. P., Bartunik H. D., Bursakov S., Calvete J., Caldeira J., Carneiro C., Moura J. J., Moura I., and Romao M. J. , Structure, Jan 15, Volume 7, Number 1, p.65-79, (1999) AbstractWebsite

BACKGROUND: The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS: The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS: The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas, Almendra, M. J., Brondino C. D., Gavel O., Pereira A. S., Tavares P., Bursakov S., Duarte R., Caldeira J., Moura J. J., and Moura I. , Biochemistry, Dec 7, Volume 38, Number 49, p.16366-72, (1999) AbstractWebsite

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein. Selenium was not detected. The UV/visible absorption spectrum of D. gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with (57)Fe and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

The solution structure of a [3Fe-4S] ferredoxin: oxidised ferredoxin II from Desulfovibrio gigas, Goodfellow, B. J., Macedo A. L., Rodrigues P., Moura I., Wray V., and Moura J. J. , J Biol Inorg Chem, Aug, Volume 4, Number 4, p.421-30, (1999) AbstractWebsite

The use of standard 2D NMR experiments in combination with 1D NOE experiments allowed the assignment of 51 of the 58 spin systems of oxidised [3Fe4S] ferredoxin isolated from Desulfovibrio gigas. The NMR solution structure was determined using data from 1D NOE and 2D NOESY spectra, as distance constraints, and information from the X-ray structure for the spin systems not detected by NMR in torsion angle dynamics calculations to produce a family of 15 low target function structures. The quality of the NMR family, as judged by the backbone r.m.s.d. values, was good (0.80 A), with the majority of phi/psi angles falling within the allowed region of the Ramachandran plot. A comparison with the X-ray structure indicated that the overall global fold is very similar in solution and in the solid state. The determination of the solution structure of ferredoxin II (FdII) in the oxidised state (FdIIox) opens the way for the determination of the solution structure of the redox intermediate state of FdII (FdII(int)), for which no X-ray structure is available.

The structure of an electron transfer complex containing a cytochrome c and a peroxidase, Pettigrew, G. W., Prazeres S., Costa C., Palma N., Krippahl L., Moura I., and Moura J. J. , J Biol Chem, Apr 16, Volume 274, Number 16, p.11383-9, (1999) AbstractWebsite

Efficient biological electron transfer may require a fluid association of redox partners. Two noncrystallographic methods (a new molecular docking program and 1H NMR spectroscopy) have been used to study the electron transfer complex formed between the cytochrome c peroxidase (CCP) of Paracoccus denitrificans and cytochromes c. For the natural redox partner, cytochrome c550, the results are consistent with a complex in which the heme of a single cytochrome lies above the exposed electron-transferring heme of the peroxidase. In contrast, two molecules of the nonphysiological but kinetically competent horse cytochrome bind between the two hemes of the peroxidase. These dramatically different patterns are consistent with a redox active surface on the peroxidase that may accommodate more than one cytochrome and allow lateral mobility.

Crystallization and preliminary x-ray analysis of a nitrate reductase from Desulfovibrio desulfuricans ATCC 27774, Dias, J. M., Bursakov S., Carneiro C., Moura J. J., Moura I., and Romao M. J. , Acta Crystallogr D Biol Crystallogr, Apr, Volume 55, Number Pt 4, p.877-9, (1999) AbstractWebsite

Periplasmic nitrate reductase from the sulfate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 contains two molybdopterin guanine dinucleotide cofactors and one [4Fe-4S] cluster as prosthetic groups and catalyzes the conversion of nitrate to nitrite. Crystals of the oxidized form of this enzyme were obtained using PEG as precipitant and belong to space group P3121 or P3221, with unit-cell dimensions a = b = 106.3, c = 135.1 A. There is one monomer of 80 kDa in the asymmetric unit, which corresponds to a Matthews ratio of 2.75 A3 Da-1. Using cryo-cooling procedures and X-rays from a rotating-anode generator, diffraction was observed to beyond 3.0 A resolution.

Enzymatic spectrophotometric determination of nitrites in beer, Girotti, S., Ferri E. N., Fini F., Ruffini F., Budini R., Moura I., Almeida G., Costa C., Moura J. J. G., and Carrea G. , Analytical Letters, 1999, Volume 32, Number 11, p.2217-2227, (1999) AbstractWebsite

A colorimetric assay for nitrite determination in beer based on c-type multiheme enzyme Nitrite reductase (NiR) isolated from Desulfovibrio desulfuricans ATCC 27774, was developed. Using the enzyme in solution, nitrite assay was linear in the 10(-8) - 10(-2) M range with a detection limit of 10(-8) M. and a recovery ranging from 90 to 107%. The imprecision ranged from 4 to 10% on the entire calibration curve. With NIR immobilised onto a nylon coil, a flow reactor was developed which showed a narrower linear range (10(-5) - 10(-2) M) and a higher detection limit (10(-5) M) than with the enzyme in solution, but made it possible to reuse the enzyme up to 100 times (50% residual activity). Sample preparation was simple and fast: only degassing and beer dilution by buffer was needed. This enzymatic assay was in good agreement with the results obtained using commercial nitrite determination kits.

Electrochemical studies on c-type cytochromes at microelectrodes, Correia dos Santos, M. M., Paes de Sousa P. M., Simões Gonçalves M. L., Lopes H., Moura I., and Moura J. J. G. , Journal of Electroanalytical Chemistry, Volume 464, Number 1, p.76-84, (1999) AbstractWebsite
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Simple and Complex Iron-Sulfur Proteins in Sulfate Reducing Bacteria, Moura, Isabel, Pereira Alice S., Tavares Pedro, and Moura José J. G. , Advances in Inorganic Chemistry, Volume Volume 47, p.361-419, (1999) Abstract
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1998
The structural origin of nonplanar heme distortions in tetraheme ferricytochromes c3, Ma, J. G., Zhang J., Franco R., Jia S. L., Moura I., Moura J. J., Kroneck P. M., and Shelnutt J. A. , Biochemistry, Sep 8, Volume 37, Number 36, p.12431-42, (1998) AbstractWebsite

Resonance Raman (RR) spectroscopy, molecular mechanics (MM) calculations, and normal-coordinate structural decomposition (NSD) have been used to investigate the conformational differences in the hemes in ferricytochromes c3. NSD analyses of heme structures obtained from X-ray crystallography and MM calculations of heme-peptide fragments of the cytochromes c3 indicate that the nonplanarity of the hemes is largely controlled by a fingerprint peptide segment consisting of two heme-linked cysteines, the amino acids between the cysteines, and the proximal histidine ligand. Additional interactions between the heme and the distal histidine ligand and between the heme propionates and the protein also influence the heme conformation, but to a lesser extent than the fingerprint peptide segment. In addition, factors that influence the folding pattern of the fingerprint peptide segment may have an effect on the heme conformation. Large heme structural differences between the baculatum cytochromes c3 and the other proteins are uncovered by the NSD procedure [Jentzen, W., Ma, J.-G., and Shelnutt, J. A. (1998) Biophys. J. 74, 753-763]. These heme differences are mainly associated with the deletion of two residues in the covalently linked segment of hemes 4 for the baculatum proteins. Furthermore, some of these structural differences are reflected in the RR spectra. For example, the frequencies of the structure-sensitive lines (nu4, nu3, and nu2) in the high-frequency region of the RR spectra are lower for the Desulfomicrobium baculatum cytochromes c3 (Norway 4 and 9974) than for the Desulfovibrio (D.) gigas, D. vulgaris, and D. desulfuricans strains, consistent with a more ruffled heme. Spectral decompositions of the nu3 and nu10 lines allow the assignment of the sublines to individual hemes and show that ruffling, not saddling, is the dominant factor influencing the frequencies of the structure-sensitive Raman lines. The distinctive spectra of the baculatum strains investigated are a consequence of hemes 2 and 4 being more ruffled than is typical of the other proteins.

Oxovanadium(IV) complexes of the dipeptides glycyl-L-aspartic acid, L-aspartylglycine and related ligands; a spectroscopic and potentiometric study, Pessoa, J. C., Gajda T., Gillard R. D., Kiss T., Luz S. M., Moura J. J. G., Tomaz I., Telo J. P., and Torok I. , Journal of the Chemical Society-Dalton Transactions, Nov 7, Number 21, p.3587-3600, (1998) AbstractWebsite

The equilibria in the systems VO2+ + L (L = Gly-L-Asp, L-Asp-Gly, N-acetyl-L-aspartic acid or succinic acid) have been studied at 25 degrees C and 0.2 mol dm(3) K(CI) medium by a combination of potentiometric and spectroscopic methods (ESR, circular dichroism and visible absorption). Formation constants were calculated from pH-metric data with total oxovanadium(Iv) concentrations of(0.6-4) x 10(-3) mol dm(-3) and ligand-to-metal (L:M) ratios of 2-8 (AspGly) or 4-15: 1 (other systems). The position of the Asp residue in the peptide chain affects the co-ordination mode of the ligands: while in the GlyAsp system bis complexes start to form at pH less than 2, for AspGly only 1 : 1 complexes form, with relatively high CD signal. The co-ordination behaviour of N-acetyl-L-aspartic and succinic acids is similar. The results of potentiometric and spectroscopic methods are self consistent. Isomeric structures are discussed for each stoichiometry proposed and the results compared with those for L-aspartic acid and dipeptides with non-coordinating side chains.

ATP sulfurylases from sulfate-reducing bacteria of the genus Desulfovibrio. A novel metalloprotein containing cobalt and zinc, Gavel, O. Y., Bursakov S. A., Calvete J. J., George G. N., Moura J. J., and Moura I. , Biochemistry, Nov 17, Volume 37, Number 46, p.16225-32, (1998) AbstractWebsite

Adenosine triphosphate sulfurylase catalyzes the formation of adenosine 5'-phosphosulfate from adenosine triphosphate and sulfate. The enzyme plays a crucial role in sulfate activation, the key step for sulfate utilization, and has been purified from crude extracts of Desulfovibrio desulfuricans ATCC 27774 and Desulfovibrio gigas. Both proteins are homotrimers [141 kDa (3 x 47) for D. desulfuricans and 147 kDa (3 x 49) for D. gigas] and have been identified, for the first time, as metalloproteins containing cobalt and zinc. EXAFS reveals that either cobalt or zinc binds endogenously at presumably equivalent metal binding sites and is tetrahedrally coordinated to one nitrogen and three sulfur atoms. Furthermore, the electronic absorption spectra display charge-transfer bands at 335 and 370 nm consistent with sulfur coordination to cobalt, and as expected for a distorted tetrahedral cobalt geometry, d-d bands are observed at 625, 666, and 715 nm. This geometry is supported by the observation of high-spin Co2+ EPR signals at g approximately 6.5.

Spectroscopic characterization of a novel tetranuclear Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans, Tavares, P., Pereira A. S., Krebs C., Ravi N., Moura J. J., Moura I., and Huynh B. H. , Biochemistry, Mar 3, Volume 37, Number 9, p.2830-42, (1998) AbstractWebsite

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2. Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.

Iron compounds after erythrophagocytosis: chemical characterization and immunomodulatory effects, Costa, L. M., Moura E. M., Moura J. J., and de Sousa M. , Biochem Biophys Res Commun, Jun 9, Volume 247, Number 1, p.159-65, (1998) AbstractWebsite

In humans, the lymphomyeloid system has a fundamental role on iron metabolism promoting its recycling due to a continuous removal of effete red blood cells. Additionally, one of the most intriguing aspects of metalloporphyrins in biology is their effect on the immune system. However, the process of erythrocyte catabolism is still poorly understood and needs further research. In the present study, we attempt to investigate the nature and the possible physiologic role of Fe compounds released after erythrophagocytosis during the removal of red blood cells. Monocyte erythrophagocytosis in vitro experiments were done to characterize chemically the Fe compounds present inside the cells and in the culture supernatants. We tested the probable immunomodulatory functions of erythrophagocytosis products over lymphocyte cultures activated in vitro with T mitogens (alpha-CD3). Data obtained from atomic absorption spectroscopy confirmed the presence of Fe in the culture supernatants of monocyte cultures after erythrophagocytosis. Also, high-spin haem complexes derived from erythrocyte catabolism were detected by electron paramagnetic electronic resonance. Finally, in vitro activated lymphocyte proliferation experiments indicate the co-mitogenic properties of monocyte culture supernatants after red blood cells phagocytosis. Thus, the results of the present work provide evidence that culture monocyte supernatants after in vitro erythrophagocytosis contain Fe (III) high-spin haem complexes and show lymphocyte proliferation co-stimulatory properties.

The surface-charge asymmetry and dimerisation of cytochrome c550 from Paracoccus denitrificans--implications for the interaction with cytochrome c peroxidase, Pettigrew, G. W., Gilmour R., Goodhew C. F., Hunter D. J., Devreese B., Van Beeumen J., Costa C., Prazeres S., Krippahl L., Palma P. N., Moura I., and Moura J. J. , Eur J Biochem, Dec 1, Volume 258, Number 2, p.559-66, (1998) AbstractWebsite

The implications of the dimeric state of cytochrome c550 for its binding to Paracoccus cytochrome c peroxidase and its delivery of the two electrons required to restore the active enzyme during catalysis have been investigated. The amino acid sequence of cytochrome c550 of Paracoccus denitrificans strain LMD 52.44 was determined and showed 21 differences from that of strain LMD 22.21. Based on the X-ray structure of the latter, a structure for the cytochrome c550 monomer from strain 52.44 is proposed and a dipole moment of 945 debye was calculated with an orientation close to the exposed haem edge. The behaviour of the cytochrome on molecular-exclusion chromatography is indicative of an ionic strength-dependent monomer (15 kDa)/dimer (30 kDa) equilibrium that can also be detected by 1H-NMR spectroscopy. The apparent mass of 50 kDa observed at very low ionic strength was consistent with the presence of a strongly asymmetric dimer. This was confirmed by cross-linking studies, which showed that a cross-linked species of mass 30 kDa on SDS behaved with an apparent mass of 50 kDa on molecular-exclusion chromatography. A programme which carried out and evaluated molecular docking of two monomers to give a dimer generated a most probable dimer in which the monomer dipoles lay almost antiparallel to each other. The resultant dipole moment of the dimer is therefore small. Although this finding calls into question the possibility of preorientation of a strongly asymmetrically charged cytochrome as it collides with a redox partner, the stoichiometry of complex formation with cytochrome c peroxidase as studied by 1H-NMR spectroscopy shows that it is the monomer that binds.

Electrochemical study on cytochrome c peroxidase from Paracoccus denitrificans: a shifting pattern of structural and thermodynamic properties as the enzyme is activated, Lopes, H., Pettigrew G. W., Moura I., and Moura J. J. G. , Journal of Biological Inorganic Chemistry, Dec, Volume 3, Number 6, p.632-642, (1998) AbstractWebsite

The di-haem cytochrome c peroxidase of Paracoccus denitrificans is a calcium binding dimer of 37.5 kDa subunits. It is responsible for reduction of H(2)O(2) to H(2)O with oxidation of cytochrome c(550) and is isolated in a fully oxidised state (inactive) in which one haem (centre I) is in a high-spin/low-spin equilibrium and high potential and the other (centre II) is low-spin and low potential. The enzyme undergoes direct electron transfer (without the need for mediators) with a 4,4'-dithiodipyridine-modified gold electrode and the response of both haem groups can be observed. By combination of the cyclic and pulse voltammetric data with the established spectroscopic information, it was demonstrated that entry of one electron to the high potential haem leads (in a mechanism involving strong haem-haem interactions) to a complex change of spin states and redox potentials of both haems in order to attain a "ready state" for binding, reduction and cleavage of the hydrogen peroxide. In the absence of endogenous calcium, haem communication can be completely disconnected and is recovered only when Ca(2+) is added, an essential step for the formation of the peroxidatic site. The intricate electrochemical behaviour of this enzyme was interpreted as a mechanism involving, both reduction and oxidation of the high potential haem, an interfacial electron transfer coupled to a homogenous chemical reaction (EC mechanism). We discuss two different models for the sequence of events leading to the appearance of the active pentacoordinated peroxidatic haem.

Metal binding to the tetrathiolate motif of desulforedoxin and related polypeptides, Kennedy, M., Yu L., Lima M. J., Ascenso C. S., Czaja C., Moura I., Moura J. J. G., and Rusnak F. , Journal of Biological Inorganic Chemistry, Dec, Volume 3, Number 6, p.643-649, (1998) AbstractWebsite

Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)(4) metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe3+ Cd2+, and Zn2+ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe3+ bound with the highest affinity, whereas the affinity of Zn2+ was greater than Cd2+ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe3+ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe3+ is reduced to Fe2+ with attendant oxidation of ligand thiols.

Isolation and characterisation of a novel sulphate-reducing bacterium of the Desulfovibrio genus, Feio, M. J., Beech I. B., Carepo M., Lopes J. M., Cheung C. W., Franco R., Guezennec J., Smith J. R., Mitchell J. I., Moura J. J., and Lino A. R. , Anaerobe, Apr, Volume 4, Number 2, p.117-30, (1998) AbstractWebsite

A novel sulphate-reducing bacterium (Ind 1) was isolated from a biofilm removed from a severely corroded carbon steel structure in a marine environment. Light microscopy observations revealed that cells were Gram-negative, rod shaped and very motile. Partial 16S rRNA gene sequencing and analysis of the fatty acid profile demonstrated a strong similarity between the new species and members from the Desulfovibrio genus. This was confirmed by the results obtained following purification and characterisation of the key proteins involved in the sulphate-reduction pathway. Several metal-containing proteins, such as two periplasmic proteins: hydrogenase and cytochrome c3, and two cytoplasmic proteins: ferredoxin and sulphite reductase, were isolated and purified. The latter proved to be of the desulfoviridin type which is typical of the Desulfovibrio genus. The study of the remaining proteins revealed a high degree of similarity with the homologous proteins isolated from Desulfovibrio gigas. However, the position of the strain within the phylogenetic tree clearly indicates that the bacterium is closely related to Desulfovibrio gabonensis, and these three strains form a separate cluster in the delta subdivision of the Proteobacteria. On the basis of the results obtained, it is suggested that Ind 1 belongs to a new species of the genus Desulfovibrio, and the name Desulfovibrio indonensis is proposed.

NMR determination of the global structure of the 113Cd derivative of desulforedoxin: investigation of the hydrogen bonding pattern at the metal center, Goodfellow, B. J., Rusnak F., Moura I., Domke T., and Moura J. J. , Protein Sci, Apr, Volume 7, Number 4, p.928-37, (1998) AbstractWebsite

Desulforedoxin (Dx) is a simple homodimeric protein isolated from Desulfovibrio gigas (Dg) containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9 kDa with 36 amino acids per monomer). In order to probe the geometry and the H-bonding at the active site of Dx, the protein was reconstituted with 113Cd and the solution structure determined using 2D NMR methods. The structure of this derivative was initially compared with the NMR solution structure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chem 1:341-353). Backbone amide protons for G4, D5, G13, L11 NH, and the Q14 NH side-chain protons, H-bonded in the X-ray structure, were readily exchanged with solvent. Chemical shift differences observed for amide protons near the metal center confirm the H-bonding pattern seen in the X-ray model (Archer M et al., 1995, J Mol Biol 251:690-702) and also suggest that H-bond lengths may vary between the Fe, Zn, and 113Cd forms. The H-bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the presence of NH-S H-bonds inferred from D2O exchange data and observed in the NMR family of structures. The presence of "H-bond mediated" coupling in Dx indicates that the NH-S H-bonds at the metal center have significant covalent character. The HSED experiment also identified an intermonomer "through space" coupling for one of the L26 methyl groups, indicating its proximity to the 113Cd center in the opposing monomer. This is the first example of an intermonomer "through space" coupling. Initial structure calculations produced subsets of NMR families with the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing toward the L26 methyl could result in a "through space" coupling. The HSED result was therefore included in the structure calculations. Comparison of the Fe, Zn, and 113Cd forms of Dx suggests that the geometry of the metal center and the global fold of the protein does not vary to any great extent, although the H-bond network varies slightly when Cd is introduced. The similarity between the H-bonding pattern seen at the metal center in Dx, Rd (including H-bonded and through space-mediated coupling), and many zinc-finger proteins suggests that these H-bonds are structurally vital for stabilization of the metal centers in these proteins.

1997
The primary structure of the split-Soret cytochrome c from Desulfovibrio desulfuricans ATCC 27774 reveals an unusual type of diheme cytochrome c, Devreese, B., Costa C., Demol H., Papaefthymiou V., Moura I., Moura J. J., and Van Beeumen J. , Eur J Biochem, Sep 1, Volume 248, Number 2, p.445-51, (1997) AbstractWebsite

The complete amino acid sequence of the unusual diheme split-Soret cytochrome c from the sulphate-reducing Desulfovibrio desulfuricans strain ATCC 27774 has been determined using classical chemical sequencing techniques and mass spectrometry. The 247-residue sequence shows almost no similarity with any other known diheme cytochrome c, but the heme-binding site of the protein is similar to that of the cytochromes c3 from the sulphate reducers. The cytochrome-c-like domain of the protein covers only the C-terminal part of the molecule, and there is evidence for at least one more domain containing four cysteine residues, which might bind another cofactor, possibly a non-heme iron-containing cluster. This domain is similar to a sequence fragment of the genome of Archaeoglobus fulgidus, which confirms the high conservation of the genes involved in sulfate reduction.

Enzymatic properties and effect of ionic strength on periplasmic nitrate reductase (NAP) from Desulfovibrio desulfuricans ATCC 27774, Bursakov, S. A., Carneiro C., Almendra M. J., Duarte R. O., Caldeira J., Moura I., and Moura J. J. , Biochem Biophys Res Commun, Oct 29, Volume 239, Number 3, p.816-22, (1997) AbstractWebsite

Some sulfate reducing bacteria can induce nitrate reductase when grown on nitrate containing media being involved in dissimilatory reduction of nitrate, an important step of the nitrogen cycle. Previously, it was reported the purification of the first soluble nitrate reductase from a sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774 (S.A. Bursakov, M.-Y. Liu, W.J. Payne, J. LeGall, I. Moura, and J.J.G. Moura (1995) Anaerobe 1, 55-60). The present work provides further information about this monomeric periplasmic nitrate reductase (Dd NAP). It has a molecular mass of 74 kDa, 18.6 U specific activity, KM (nitrate) = 32 microM and a pHopt in the range 8-9.5. Dd NAP has peculiar properties relatively to ionic strength and cation/anion activity responses. It is shown that monovalent cations (potassium and sodium) stimulate NAP activity and divalent (magnesium and calcium) inhibited it. Sulfate anion also acts as an activator in KPB buffer. NAP native form is protected by phosphate anion from cyanide inactivation. In the presence of phosphate, cyanide even stimulates NAP activity (up to 15 mM). This effect was used in the purification procedure to differentiate between nitrate and nitrite reductase activities, since the later is effectively blocked by cyanide. Ferricyanide has an inhibitory effect at concentrations higher than 1 mM. The N-terminal amino acid sequence has a cysteine motive C-X2-C-X3-C that is most probably involved in the coordination of the [4Fe-4S] center detected by EPR spectroscopy. The active site of the enzyme consists in a molybdopterin, which is capable for the activation of apo-nit-1 nitrate reductase of Neurospora crassa. The oxidized product of the pterin cofactor obtained by acidic hidrolysis of native NAP with sulfuric acid was identified by HPLC chromatography and characterized as a molybdopterin guanine dinucleotide (MGD).

Fe-57 Q-band pulsed ENDOR of the hetero-dinuclear site of nickel hydrogenase: Comparison of the NiA, NiB, and NiC states, Huyett, J. E., Carepo M., Pamplona A., Franco R., Moura I., Moura J. J. G., and Hoffman B. M. , Journal of the American Chemical Society, Oct 1, Volume 119, Number 39, p.9291-9292, (1997) AbstractWebsite
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